T-cell Receptor Beta Chain Research Articles

Abstract 2272: Exploratory analysis of T cell repertoire dynamics upon systemic treatment with the oncolytic virus pelareorep in combination with pembrolizumab and chemotherapy in patients with advanced pancreatic adenocarcinoma

Abstract Background: Pelareorep is an immuno-oncolytic virus that induces an inflamed tumor phenotype in metastatic adenocarcinoma of the pancreas (MAP). Systemically delivered pelareorep in combination with chemotherapy achieves 1 & 2 year-survival rates of 46% & 24% in MAP pts, respectively. Analysis of tumor tissue from patients (pts) treated with pelareorep, chemotherapy and anti-PD-L1 have shown reovirus RNA and protein replication, T-cell infiltration, and upregulation of PD-L1, highlighting that effective T-cell recognition of tumor antigens may be critical to success for this combination therapy. Thus, we hypothesized that pelareorep in combination with chemo and pembrolizumab in pts with MAP would alter the peripheral T-cell repertoire, creating new T-cell clones via the release of novel neoantigens in addition to expanding existing T-cell clones. Methods: A phase 1b study enrolled 11 MAP pts who progressed after first-line treatment. Pts received pelareorep (4.5e10 TCID 50 IV, D1 & D2), plus pembrolizumab (2mg/kg IV, D8) plus either 1) 5-FU (LV (200 mg/m2 /5-FU 200 mg /m2 IV bolus, 5-FU 1200mg/m2 continuous IV infusion D1) or 2) gemcitabine (1000 mg/m2 IV, D1), or 3) irinotecan (125 mg/m2 IV, D1) q3w, until disease progression/unacceptable toxicity. DNA from peripheral blood mononuclear cells from nine patients at cycle 1 day 1 (C1D1) & C2D1 (approx. 3 weeks later) was analyzed using the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle) sequencing the T-cell receptor beta chain region to interrogate changes in the T-cell repertoire. Results: The median Morisita index between C2D1 and C1D1 was 0.83 with 3 samples below 0.6, indicative of significant peripheral repertoire turnover. The median number of expanded clones equated to 45.7 per 100,000 cumulative templates; normal variation over 4 weeks is ~ 5-10 expanded clones. Strikingly, most (median: 86%) peripheral clonal expansion occurred in clones below the limit of detection at C1D1. Cox regression analysis revealed that high peripheral clonality correlates with progression free survival at C1D1 (p=0.01, HR=0.053). Moreover, high clonality correlates with overall survival at both C1D1 (p=0.013, HR=0.124) and C2D1 (p=0.010, HR=0.079). Conclusions: High levels of peripheral T-cell repertoire turnover occur between C1D1 and C2D1. Repertoire turnover is accompanied by significant clonal expansion, mostly by expansion of new clones (i.e. undetected in C1D1). Higher peripheral clonality is associated with better progression free survival at C1D1, and overall survival at C1D1 and C2D1. This research highlights the potential utility of T-cell clonality as a predictive and prognostic biomarker to pelareorep therapy and warrants further clinical investigation. Citation Format: Grey A. Wilkinson, Devalingam Mahalingam, Sukeshi Patel Arora, Paul A. Fields, Patrick Raber, Karol Cheetham, Matt Coffey. Exploratory analysis of T cell repertoire dynamics upon systemic treatment with the oncolytic virus pelareorep in combination with pembrolizumab and chemotherapy in patients with advanced pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2272.

Features and therapeutic potential of T-cell receptors in high-grade glioma.

Previous studies have shown that endogenous T cells play an important role in the prolonged survival time of high-grade glioma (HGG) patients. Our objectives were to investigate the features of T-cell receptor (TCR) repertoires in HGG patients and to elucidate any potential therapeutic value. During November 2011 and December 2018, tumor tissues and blood samples of 35 patients with HGG who underwent surgery at Beijing Tiantan Hospital or Beijing Shijitan Hospital were selected after surgery. After isolating DNA from samples, multiple rounds of PCR were performed to establish a DNA immune repertoire (IR). Then, the sequences and frequencies of the complementarity-determining 3 (CDR3) region in TCR beta chain (TRB) were identified by high-throughput sequencing and IR analysis. A survival follow-up was conducted monthly thereafter until December 2018. Finally, the t test and Mann-Whitney test were used to compare statistical differences between two sets of data. The Shannon diversity index (SHDI) of TRB sequences of HGG patients was significantly lower than that of healthy individuals (7.34 vs. 8.45, P = 0.001). The SHDI of TRB sequences of glioblastoma (GBM) patients with more than 16 months survival time was much higher than that of GBM patients with shorter survival times in both tumor tissues (3.48 ± 0.31 vs. 6.21 ± 0.33, t = -5.49, P = 0.002) and blood cells (6.02 ± 0.66 vs. 7.44 ± 0.32, t = -2.20, P = 0.036). In addition, patients achieved a distinctly higher proportion compared to that of healthy individuals in the proportion of TRBV9 and TRBV5 functional regions (9.83% vs. 6.83%, P = 0.001). Surgical tissue from patients who survived more than 16 months yielded a much higher proportion of TRBV4 and TRBV9 regions (7.14% vs. 3.28%, t = 3.18, P = 0.019). In surgical tissues from two GBM patients who survived for longer than 46 months, we found a potentially therapeutic TCR sequence. HGG patients have less species diversity of TCR repertoires compared with that of healthy individuals. TRBV9 regions in TCRs may be protective factors for long-term survival of GBM patients.

Clonal expansion of tumor infiltrating lymphocytes (TILs) in the peripheral blood of metastatic melanoma patients is significantly associated with response to CTLA4 blockade-based immunotherapy.

2541 Background: Patients with metastatic melanoma were treated on a clinical trial with tremelimumab and High Dose Interferon-Alfa (HDI) (Tarhini. J Clin Oncol. 2012). We previously reported that patients who achieved disease control and clinical response had significantly greater T-cell clonality (p = 0.0008) and T-cell fraction (p = 0.044) respectively in their pretreatment tumor biopsy samples (Tarhini. J Clin Oncol. 2017). In this study, we further characterize T-cell repertoire clonality and clonal expansion in the peripheral blood at different time points to evaluate the association between repertoire features and clinical response. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks and HDI was given concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 33) were obtained at baseline, day 29, and day 85 (following tremelimumab-HDI treatment); tumor samples at baseline were also obtained (N = 18). The T-cell receptor beta chain (TCRB) repertoire of PBMCs and tumor samples was immunosequenced using the immunoSEQ assay (Adaptive Biotechnologies), and repertoire clonality was assessed at baseline, day 29, and day 85. Differential abundance analysis was used to detect and quantify peripheral clonal expansion pre- versus post-treatment and identify the subset of peripheral clones also detected in the tumor repertoire. The Morisita Index of repertoire similarity was also calculated to compare global repertoire changes between pre- and post-treatment PBMC samples. Results: T-cell repertoire turnover, as measured by the Morisita Index, showed a trend towards responders (CR/PR) having greater turnover (lower Morisita Index) post-treatment than non-responders (SD/PD). Similarly, the total number of clones expanding in the peripheral repertoire varied over time within an individual (p = 0.034) but was not significantly affected by response to therapy (p = 0.275) or by on-treatment time point (p = 0.768). When the analysis was restricted to peripherally expanded clones that were also found in the tumor repertoire, responders had significantly more TILs expanded in the periphery at day 29 than non-responders (p = 0.036). Conclusions: Our analysis of the peripheral T-cell repertoire following treatment showed that detection of TILs in early peripheral clonal expansion correlates with response to therapy.

Human urothelial bladder cancer generates a clonal immune response: The results of T-cell receptor sequencing

BackgroundHigh T-cell receptor (TCR) repertoire clonality is associated with clinical response to immune checkpoint blockade in bladder cancer. ObjectiveTo determine if TCR repertoire is more clonal in tumors than in benign inflammation. MethodsWe prospectively identified 12 patients with bladder lesions undergoing transurethral resection. Specimens were collected at time of transurethral resection and stored at −80C. DNA was extracted and high throughput DNA sequencing of the CDR3 region of the TCR beta chain using the immunoSEQ assay (Adaptive Biotechnologies) was performed. T-cell fraction, clonal dominance, and maximum frequency of TCR clone were assessed. ResultsOf the 12 bladder lesions resected, 3 of 12 were cT0, 3 of 12 were cTa, 3 of 12 were cT1, and 3 of 12 were cT2 or greater. The median number of T cells in urothelial carcinoma specimens (UC+) and benign (UC−) specimens was 5,569 and 25,872, respectively. The number of unique TCRs sequenced in UC+ and UC− specimens was 3,069 and 9,680, respectively. The median tumor infiltrating lymphocyte percentage in UC+ and UC− specimens was 2% and 12%, respectively. The UC+ specimens demonstrated clonality as evidenced by identification of a specific T-cell clone being present in up to 17% of the total tumor infiltrating lymphocyte pool, in contrast to 2% among UC− specimens. ConclusionsPrimary urothelial tumors contain clonally expanded T-cell populations. These data support the hypothesis that bladder tumors induce an antigen-driven immunogenic host response, in contrast to the benign inflammatory response, which does not appear to demonstrate any T-cell clonal dominance.

A novel method for high throughput TCR single cell VDJ-pairing with phenotypic analysis

Abstract Single cell transcriptional analysis reveals a wealth of information that is obscured when analyzing the RNA of a sample en masse. Furthermore, it is important to know the identity of the TCR chain VDJ-rearrangement associated with the phenotypic state. This information can provide direct calculations of clonal frequency in various cell subsets, tracking of specific lymphocytes with treatment, and reveal paired information for both chains of the receptor for downstream Car-T development. Here, we discuss a novel method that identifies paired VDJ-rearrangements for both TCR alpha and beta chains from single cells barcoded via the BD Rhapsody Express single cell system. The phenotype of the cell is obtained using the BD Rhapsody RNA-seq kit, while the receptor information is amplified from the same cDNA using an iRepertoire-derived method incorporating a multiplex mix of primers associated with both the TCR alpha and beta loci. This targeted-seq strategy requires less read depth when compared to template-switch approaches. In one experiment, 3,000 TCR-beta unique CDR3 (uCDR3) were identified from ~7,000 barcoded cells using 1.5 million NGS reads with a 0.83 Pearson correlation upon repeat. Approximately 1,000 TCR alpha-beta paired uCDR3 were also called from the data. Additionally, the BD phenotyping results independently verify the immune repertoire data since the TCR beta chain constant gene overlays with the TCR-beta uCDR3 clonotypes for CD4+ and CD8+ subsets but is largely absent in the CD14+ monocyte population for both analyses. Thus, the developed technology provides the ability to simultaneously pair both alpha and beta chains for thousands of T-cells while obtaining information related to the functional status of each cell.

Physical cognate pairing of mouseTCR alpha-beta VDJ-rearrangements with phenotype directly from FACS sorted cells

Abstract Phenotypic characterization of single cells paired with immune receptor information can provide valuable insight into T cell function, which may be beneficial for the development of personalized therapies. In order to comprehensively analyze T cell function and specificity, it is necessary to integrate antigen receptor sequencing with phenotypic markers at the single cell level. We therefore developed a sensitive technology called iPair+™ that enables the simultaneous amplification and sequencing of paired mouse TCR alpha and beta chains with functional gene markers in a single, sorted immune cell via a combination of flow cytometry, multiplex PCR, and next generation sequencing. We utilized iPair+™ to study CD4 and CD8 T cells isolated from treated and non-treated mice from a breast cancer model. CDR3 diversity, clonality, and frequency were analyzed together with 80+ phenotypic genes. When cells isolated from both treated and non-treated mice were compared, we found gene expression levels changed significantly with treatment. Our data demonstrates that genes that were upregulated prior to treatment were associated with cancer growth, while genes upregulated after treatment were associated with T cell activation and function in cancer repression or growth inhibition. Our study indicates that iPair+ technology could reveal important insights into T cell function, clonal development, and immunotherapy response during treatment.

Deep targeted sequencing reveals the diversity of TRB-CDR3 repertoire in patients with preeclampsia

Preeclampsia (PE) is one of the major causes of maternal and perinatal mortality worldwide. This study aimed to determine the immunological characteristics of PE patients and normal pregnancy at the T cell receptor beta-chain (TRB) level by using high-throughput sequencing. High-throughput sequencing was performed to analyze the expression of TRB-CDR3 in circulating T cells. T cells were isolated from 36 healthy pregnant women, 24 patients with severe PE, and 18 patients with moderate PE. Rearranged mRNA sequences were assigned to their germline V, D, and J counterparts, and translated into proper amino acids by the IMGT database. In general, PE samples had more TRB-CDR3 reads and types than those of normal pregnant woman in the circulation, but the mean number of TRB-CDR3 reads and unique TRB-CDR3 reads in severe group was lower than that in the moderate group. In PE patients, the V7_9 and V20_1 gene loci were more prevalent than in healthy pregnant women. In addition, 4 kinds of TRB-CDR3 peptides were found to be highly relevant to the pathogenesis of PE. Of them, peptides matched to herpes simplex virus antigen-specific T cells were much lower in PE samples.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Tasmanian devils with contagious cancer exhibit a constricted T-cell repertoire diversity

The Tasmanian devil (Sarcophilus harrisii) is threatened by a contagious cancer, known as Devil Facial Tumour Disease (DFTD). A highly diverse T-cell receptor (TCR) repertoire is crucial for successful host defence against cancers. By investigating TCR beta chain diversity in devils of different ages, we show that the T-cell repertoire in devils constricts in their second year of life, which may explain the higher DFTD prevalence in older devils. Unexpectedly, we also observed a pronounced decline in TCR diversity and T cell clonal expansion in devils after DFTD infection. These findings overturned the previous assumption that DFTD did not directly impact host immunity.

Abstract B024: Using single-cell paired sequencing to isolate cancer-specific T-cell receptors for cancer immunotherapy

Abstract Immunocore’s ImmTAC™ (Immune Mobilising Monoclonal TCR Against Cancer) platform combines affinity-enhanced T-cell receptor (TCR)-based targeting with an anti-CD3 scFv effector function to activate a cytotoxic T-cell response against cancer cells. A key part to this process is the identification of tumour epitope specific TCRs from tumor antigen-reactive T-cells. Here, we describe an integrated in-house process leading to the isolation of TCRs specific for validated cancer epitopes, coupled with rapid identification of TCR chains from individual clones using single cell sequencing. The process involves first strand cDNA generation and universal amplification using SmartSeq2 chemistry, followed by targeted sequencing of the TCR alpha and beta chains using next-generation sequencing (NGS). We have also leveraged the 10x Genomics VDJ/5’ counting platform to label and pool multiple experimental clones for repertoire sequencing within a single run. Together with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq), we can reliably assign each T-cell clone to its sample of origin paired with transcriptomic information of epitope specific T-cell populations, linking TCR sequences to their functional phenotype. Citation Format: Karolina Lech, Lucia Correia, Max Beckmann, Maria Busz, Sean Collison, Sterenn Davis, Paraskevi Mallini, Sarah Scaife, Joseph Dukes, Bent K. Jakobsen, Luke Williams, Michelle Teng. Using single-cell paired sequencing to isolate cancer-specific T-cell receptors for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B024.

Abstract A043: Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro

Abstract Overall survival of pediatric B-precursor ALL patients reached 90% in recent years. However, the outcome for refractory or relapsed children remains very poor. Anti-CD19 chimeric antigen receptor T-cells (CD19-CAR) showed significant antileukemic activity in relapsed and refractory B-precursor ALL. Especially in children, isolation of a suitable T-cell amount for autologous CAR T-cell manufacturing can be challenging due to low blood volume, low T-cell counts and clinical condition. In this case, the adoptive transfer of CAR T-cells from an unmatched healthy third-party donor provides a promising strategy. In order to prevent life-threatening graft-versus-host disease, a knockout (KO) of the endogenous T-cell receptor (TCR) has to be performed. Here, we generated CD19-CARs with a CRISPR/Cas9 mediated TCR KO, which remain highly functional and show strongly reduced alloreactivity compared to conventional CAR T-cells introduced into third-party T-cells. T-cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and activated via anti-CD3/anti-CD28 stimulation. Retroviral transduction of a second generation anti-CD19 CAR (containing CD3zeta and 4-1BB stimulatory domains) was performed, followed by CRISPR/Cas9 mediated KO of the T-cell receptor beta chain via electroporation. After eleven days of expansion in the presence of IL-7 and IL-15, cells were purified for TCR KO-CD19-CAR T-cells via magnetic separation. Finally, the cell product was analyzed for cellular characteristics, functionality and alloreactivity by flow cytometry. A mean transduction rate of 37% for CD19-CARs and 40% for TCR KO-CD19-CARs was reached as well as a mean TCR KO rate of 78%. Both CD19-CARs as well as TCR KO-CD19-CARs showed suitable amounts of CD4- (45% vs. 33%) and CD8-T-cells (37% vs. 48%). The phenotype of CD19-CARs and TCR KO-CD19-CARs were comparable with mainly central memory (CM) (38% vs. 40%) and effector memory (EM) (57% vs. 51%) T-cells. The expansion of TCR KO-CD19-CARs was significantly reduced compared to conventional CD19-CARs (54-fold vs. 109-fold). This effect was not mediated by the loss of the TCR, but due to electroporation procedure. While CD19-CARs with or without TCR KO showed almost no background expression of the activation marker CD25 (2% vs 1%), contact with CD19-expressing targeT-cells resulted in a comparable upregulation of CD25 in both groups (95% vs. 94%). Co-culture with a CD19-expressing targeT-cell line led to an increased Interferon-γ secretion compared to unstimulated CARs, which was not significantly altered by the TCR KO (17% CD19-CAR vs. 14% TCR KO-CD19-CAR). CD19-dependent proliferative capacity of CAR T-cells was not influenced by loss of the TCR, as in both cases 97% of the T-cells proliferated after antigen recognition. Both CD19-CARs as well as TCR KO-CD19-CARs showed high, antigen-specific killing of 86% vs. 87% of the CD19-expressing targeT-cells at a 1:1 effector to target ratio. To evaluate the alloreactive potential of those T-cells, T-cells were co-cultured with irradiated PBMCs pooled from six different donors. 20% of TCR-expressing T-cells showed proliferation upon contact with non-HLA-matched PBMCs, whereas T-cells with a TCR KO showed almost no proliferation (<3%), demonstrating significantly reduced alloreactivity of TCR KO T-cells. CD19-CAR T-cells lacking the endogenous TCR show a balanced CD4 to CD8 ratio and high proportion of the favorable CM T-cell phenotype. TCR KO-CD19-CARs remain highly functional and show similar activation, cytotoxicity, proliferative capacity and cytokine secretion as conventional CD19-CARs upon antigen recognition. T-cells with TCR KO do not mediate an alloreactive response to non-HLA-matched PBMCs and therefore are promising candidates for the generation of CAR T-cells derived from nonmatched healthy third-party donors or for use of donor-derived CAR T-cells after haploidentical stem cell transplantation. Citation Format: Dana Stenger, Tanja Stief, Theresa Käuferle, Semjon Manuel Willier, Felicitas Rataj, Kilian Schober, Ramin Lotfi, Beate Wagner, Dirk H. Busch, Sebastian Kobold, Franziska Blaeschke, Tobias Feuchtinger. Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A043.

Multiomic Insights into Novel Treg Subset

Using epigenetic reprogramming through Somatic Cell Nuclear Transfer, we developed two regulatory T cell (Treg) models, T138 (Ku M., et al., PNAS 2016) and T143 (Ku M., et al., JACI 2018). While both Treg models were thymically derived, T143 displayed characteristics of agonist-selection (aTreg), which had been postulated for all Treg cells. However, T138 was quite distinct with a higher degree of interaction between TCR beta-chain and MHC-II rather than TCR and agonist. We refer to as T138 as natural-occurring Treg cell (nTreg). Strikingly, we identified FoxP3-negative T cells expressing the same TCR as nTreg and aTreg. Especially, FoxP3-negative T cells isolated from T138 were heavily poised in their ability to differentiate into FoxP3-expressing cells rather than Th1-differentation.In order to further identify new transcription factors that mediated this epigenetically poised state in FoxP3-negative T138 (pre-nTreg) cells and to identify accessible promoters and enhancers that are associated with the pro-FoxP3 poised state, we performed ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing). We identified several loci that are more accessible in pre-nTreg cells than in wild-type (WT) cells. Motif analysis revealed enrichment of several lineage-determining transcription factor binding motifs, such as Atf3. Strikingly, transcriptomic analysis demonstrated that Atf3 is specifically expressed in T138 and not in WT. Interestingly; it has previously been shown that the FoxP3-locus contains an Atf-binding site, suggesting an important link of pre-nTreg transcriptional programs to its development into nTreg.Since ATAC-Seq signals show similar enrichment at both active and poised (bivalently-marked by both H3K4me2 and H3K27me3 marks) promoters, we performed H3K4me2 and H3K27me3 chromatin-immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) to further identify poised promoters, which gear e.g. pre-nTreg towards a FoxP3+ cellular fate.In order to identify unique phosphorylation sites during activation of pre-nTreg and nTreg cells, we analyzed the phospho-proteome. We identified several differential phosphorylated peptides. Particularly, we found about 100 peptides uniquely phosphorylated in FoxP3- pre-nTreg cells. Together with the transcriptional analysis this clearly demonstrates the unique characteristics of this novel pre-nTreg population.Several mechanisms had been shown to mediate immune regulatory function of Treg cells. Strikingly, we identified Ctla4 and Lag3 to be differentially expressed in T138, T143 and the Treg pool in WT mice. This is of particular interest since, e.g. CTLA4 is currently being explored for immunotherapy. Differential expression of Ctla4 provides evidence for selective modifications of a Treg subset through anti-CTLA4 treatment. Our data supports the concept that anti-CTLA4 treatment might function through targeting of a tumor-infiltrating Treg subset rather than check point inhibition.Taken together our multiomics and functional data on Treg cells clearly demonstrated the existence of distinct Treg subsets, which has major implications in better understanding disease mechanisms and in devising new future therapies. DisclosuresSabouri-Ghomi:Fate Therapeutics: Employment.

Adoptive T-Cell Therapy with TCL1-Specific TCR for B-Cell Lymphomas

Chimeric antigen receptor (CAR)-modified T-cell therapy targeting CD19 induces high response rates in patients with relapsed or refractory B-cell lymphomas. However, about 60% of patients experience primary or secondary resistance after CD19-targeted CAR T-cell therapy and a major of cause of failure appears to be due to loss of CD19 expression on the tumor. Therefore, novel targets for adoptive T-cell therapeutic approaches are needed to further improve clinical outcome in these patients.T-cell leukemia/lymphoma antigen1 (TCL1) is an oncoprotein that is overexpressed in multiple B-cell malignancies including follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL). Importantly, it has restricted expression in only a subset of B cells among normal tissues. We previously identified a TCL1-derived HLA-A2-binding epitope (TCL170-79 SLLPIMWQLY) that can be used to generate TCL1-specific CD8+ T cells from peripheral blood mononuclear cells of both HLA-A2+ normal donors and lymphoma patients. More importantly, we showed that the TCL1-specific CD8+ T cells lysed autologous primary lymphoma cells but not normal B cells (Weng et al. Blood 2012).To translate the above discovery into clinic, we cloned the T-cell receptor (TCR) alpha and beta chains from a TCL1-specific CD8+ T-cell clone and showed that this TCL1-TCR could be transduced into polyclonal donor T cells using a lentiviral system with a transduction efficiency of >40% as determined by TCL170-79 tetramer positive T cells. Furthermore, we demonstrated that the TCL1-TCR-transduced T cells recognized T2 cells pulsed with TCL170-79 peptide producing IFN- γ >8 ng/ml and IL-2 >350 ng/ml but were not reactive to control HIV-Gag peptide (IFN- γ <0.1 ng/ml and IL-2 <0.2 ng/ml). The TCL1-TCR-transduced T cells recognized TCL170-79 peptide pulsed onto T2 cells at a concentration of 1-10 nM (IL-2 >10 ng/ml) suggesting it has moderate to high avidity. Importantly, TCL1-TCR-transduced T cells lysed HLA-A2+ (up to 43% lysis of Mino and 25% lysis of Jeko-1 at 40:1 Effector:Target ratio) but not HLA-A2- lymphoma cell lines (5.5% lysis of HLA A2- Raji and 2.3% lysis of Daudi at 40:1 Effector:Target ratio). TCL1-TCR-transduced T cells were also cytotoxic to HLA-A2+ primary lymphoma tumor cells (up to 48% lysis of CLL, 43% lysis of FL, 41% lysis of DLBCL, 46% lysis of splenic marginal zone lymphoma, and 11% lysis of MCL at 40:1 Effector:Target ratio) but not normal B cells derived from the same patients. Lastly, TCL1-TCR transduced T cells showed high efficacy in in vivo models. Adoptive transfer of the TCL1-TCR-tranduced T cells significantly reduced lymphoma tumor growth and extended survival in Mino mantle cell lymphoma cell line xenograft model (48% survival in TCL1-TCR-T treated group vs. 12.5% survival in control group at 10 weeks n=7-8 mice/group; P=0.02).Collectively, our data suggest that the high expression in B-cell tumors, restricted expression in normal tissues, and presence of an immunogenic CD8 T-cell epitope, make TCL1 a target for T cell-based therapeutic approaches in multiple B-cell malignancies. Our results also demonstrate that the TCL1-specific TCR-transduced T cells may serve as a novel adoptive immunotherapy approach for the treatment of patients with various B-cell malignancies (including FL, MCL, DLBCL, CLL).Acknowledgments: This study is supported by MD Anderson Moon Shot Program and CPRIT and the National Natural Science Foundation of China Grant (No. 81570189) DisclosuresNeelapu:Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Karus: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Somatic Mutations in T Cells As Possible Regulators of Immunodeficiency

▪The pathogenesis of common variable immunodeficiency (CVID) and many other immunodeficiencies is elusive, and no cure for these diseases exists. The characteristic features of CVID are immunoglobulin deficiency and recurrent infections, but autoimmunity co-manifests in 30% and lymphoproliferation in 50% of CVID cases. The failure to produce sufficient quantities of immunoglobulins is attributed to B cells, but as T cells are critical players in adaptive immune response and autoimmune disease, they have also suggested to play a role in CVID. As monogenic germline mutations account only for 2-10% of CVID cases, the etiology of CVID remains unknown in most cases.To study if somatic mutations in T cells contribute to immunodeficiencies, we recruited patients with broad immune dysregulation: 8 patients with late-onset CVID, and 9 patients with other type of immunodeficiency and/or severe autoimmunity. All patients signed informed consent and the declaration of Helsinki principles were followed. Study design and key patient characteristics are depicted in the Figure. To discover somatic mutations in T cells, we sequenced both CD4+ and CD8+ cells with a custom gene panel comprising of 2500 immune-related genes with an average coverage of 500x. Somatic variants were identified using the GATK MuTect2 toolset by using a panel of 21 healthy controls' CD4+ and CD8+ cells as a background. Variants were called using paired samples (CD4+ vs CD8+ and vice versa). This approach identifies variants that have occurred in mature T cells. To complement this approach, we performed variant calling also in single-sample mode to identify variants originating from hematopoietic progenitors.Paired-sample analyses revealed 44 somatic mutations in mature T cells in 10/17 (59%) patients: 30 (68%) in CD8+ and 14 (32%) in CD4+ cells. The mutations included 2 frameshift-, 37 missense-, and 5 nonsense variants. In silico tools (both Polyphen-2 and SIFT) predicted 19 (51%) of the missense mutations to be damaging. The Catalogue Of Somatic mutations In Cancer (COSMIC) database included 9/44 (20%) of all mutations. Also, pathway analysis annotated 20% of the mutated genes to be involved in inflammatory response regulation, 27.5% in protein phosphorylation regulation, and 22.5% in positive regulation of cell proliferation. Interesting mutation findings included two patients with STAT5B mutations (N418K and T628S) with a low variant-allele (VAF) frequencies (3.2-3.6% in CD4+ cells), one patient with a KRAS T58I mutation (VAF 7.9% in CD8+), and two patients with distinct C5AR1 mutations (R197W and T62M). Complementing the paired-sample variant calling strategy, single-sample analyses revealed mutations associated with clonal hematopoiesis with low VAFs (2.2-5.5%) in T cells in 4 (23.5%) patients. Three patients had DNMT3A mutations and one patient 4 distinct TET2 mutations (2 nonsense-, 1 frameshift-, and 1 missense mutations).CD4+ and CD8+ T-cell receptor (TCR) repertoires were profiled with deep TCR beta chain (TCRB) sequencing. Healthy controls' CD4+ and CD8+ cells (n=27) were used as comparators. Although some CVID patients harbor major (17-20% of CD8+) T-cell clones, overall CD4+ or CD8+ clonality did not significantly differ from age-matched healthy controls. As a marker of selective pressure, CVID patients' CD4+ and CD8+ cells harbored more clones that shared a CDR3 amino-acid but had distinct nucleotide CDR3 sequences than healthy controls (7.0% vs 3.3% of all amino-acid rearrangements, p=0.021 for CD8+; 4.3% vs 3.5% p=0.013 for CD4+). These TCRs were not enriched with pathogen-specific (such as cytomegalovirus or Epstein-Barr virus) TCRs. Moreover, high CD4+ clonality was associated with a lower frequency of switched-memory-B cells.In conclusion, somatic mutations in T cells occurred in 59% of patients with immunodeficiency in this study, and some of the mutated genes (such as STAT5B) have previously been implicated for the pathogenesis of autoimmunity and lymphoproliferation. Also, clonal hematopoiesis in T cells was discovered in 23.5% of patients. The overall T-cell clonality was not increased, but CVID patients showed slight selective pressure in the TCR repertoire. Our results demonstrate that further research on somatic mutations in immunodeficiencies is needed, as they may contribute to disease pathogenesis in a subset of immunodeficiency patients. DisclosuresMartelius:Gilead: Other: lecture fee; Octapharma: Other: travel grant; CSL Behring: Other: lecture fee and travel grant; MSD: Other: lecture fee and travel grant; Sanguin: Other: travel grant and grants. Kankainen:Medix Biochemica: Consultancy. Seppänen:CSL Behring: Other: Chairing and speaker fees. Mustjoki:Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding.

High-Throughput Sequencing of the T-Cell Receptor Beta Chain Gene Repertoire in Chronic Lymphocytic Leukemia.

High-throughput, next-generation sequencing (NGS) offers a unique opportunity for in-depth characterization of adaptive immune receptor repertoires. Nevertheless, limitations and pitfalls exist in every step of both the experimental and the analytical procedure, leading to discrepancies in the literature and incomprehensive and/or altogether misleading results. Thus, standardization of protocols in NGS immunogenetics is urgently needed.Here, we describe the experimental protocol that we developed for T-cell receptor beta chain (TRB) gene repertoire analysis in chronic lymphocytic leukemia, aiming to provide a reproducible and biologically meaningful output. Although optimized for TRBV-TRBD-TRBJ gene rearrangements, this protocol may be customized for other adaptive immune receptor sequences, as well.

1163P - Study on treatment of stage IV solid tumors with mutant neoantigen specific T cells

Background: As an important tumor immunotherapy, the specificity and efficiency of PD1 inhibitor is not yet satisfactory. The treatment of solid tumor with mutant neoantigen specific T (MNaS-T) cells developed in this study is an adoptive cell therapy which is specific for each patient. The aim is to explore the difference in safety and efficacy between MNaS-T cells and PD1 inhibitors, and to evaluate the charateristic of immune repertoire (IR) as predictive biomarker.Table: 1163PDemographic and clinical characteristics of 22 patientsCharacteristicMNaS-T cells plus PD1 inhibitors plus BSC (N = 11)PD1 inhibitors plus BSC (N = 11)Disease Progression (N=4)No Disease Progression (N=7)Total (N = 11)Age--no.(%)<604 (100)5 (71.4)9 (81.8)6 (54.5) > =6002 (28.6)2 (18.2)5 (45.5)Sex--no.(%)Male3 (75)3 (42.9)6 (54.5)8 (72.7)Female1 (25)4 (57.1)5 (45.5)3 (27.3)Previous treatments--no.Chemotherapy471111Targeted therapy471111ECOG--no.(%)0000011676≥23145Stage--no.IV471111Peripheral IR Diversity--mean (SD)EvennessBaseline0.01 (0.01)0.07 (0.08)0.05 (0.07)1st cycle0.05 (0.05)0.06 (0.07)0.05 (0.06)1st cycle / Baseline3.29 (2.09)0.85 (0.44)1.74 (1.71)ClonalityBaseline0.32 (0.09)0.25 (0.18)0.27 (0.15)1st cycle0.25 (0.12)0.27 (0.16)0.26 (0.14)1st cycle / Baseline0.76 (0.18)1.2 (0.27)1.04 (0.32) Open table in a new tab Methods: A total number of 11 patients with advanced solid tumors who failed after multiline treatments were recruited. They were treated with MNaS-T cells, PD1 inhibitors and BSC; other 11 patients were treated with PD1 inhibitors and BSC as control. Peripheral blood was collected at baseline and per cycle (21-28d) respectively. Multiple PCR and NGS on TCR beta chain was used to detect IR. Results: PFS of two groups had a statistical significance (P < 0.05), suggesting MNaS-T cells prolong patients' PFS. The safety was analyzed from routine blood urine stool test, coagulation function, liver and kidney function. There was no significant difference at baseline (P > 0.05). Compared with C group, total protein and albumin in T group had a transient decrease in 3rd, 4th and 5th follow-up respectively (P < 0.05), however, It can be recovered autonomously before 6th cycle. Evenness index and Clonality indexe were examined to illuminate the diversity and clonality of IR seperately. Compared to baseline, T cell repertoir of disease-progression patients and no-disease-progression patients after 1st cycle showed significantly different changes: Evenness 3.29 vs 0.85, P = 0.013; Clonality 0.76 vs 1.20, P = 0.015. Elevated Clonality may indicate amplification of tumor specific T cells which could recognize mutant neoantigen specifically. Conclusions: The combined immunotherapy of MNaS-T cells and PD1 inhibitors is more effective than PD1 inhibitor alone in prolonging the PFS, and has a good safety. IR Clonality change shows its potential as a predictive biomarker. Legal entity responsible for the study: J. Shunchang. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

66P - TCR beta chain convergence defines the tumor infiltrating T cell repertoire of melanoma and non-small cell lung carcinoma

66P - TCR beta chain convergence defines the tumor infiltrating T cell repertoire of melanoma and non-small cell lung carcinoma

Abstract A33: Tumor derived T cell clones for evaluation of check point inhibitor therapeutics

Abstract Improving clinical outcomes using immunotherapeutics in cancer treatments will depend on a variety of factors including the composition and phenotype of effector T cell in tumors, the inhibitory factors from myeloid, tumor and stromal compartments of the tumor and selection of efficacious combination of therapeutic immunomodulatory agents that either neutralize immune checkpoint inhibitors or activate T cell costimulatory pathways. To address the issue of selecting optimal combinations of immune modulating agents we developed an in-vitro culture system for allo-stimulating and expanding tumor infiltrating lymphocytes (TILS) from tumor digests with an MHC Class II alloantigen expressing cell line. We selected allo-specific CD4 and CD8 clones from expanded TILS with an exhausted phenotype confirmed by cell surface expression of PD-1, LAG3 and TIGIT. T cell clones were incubated with the alloantigen expressing cell lines transduced with different combinations of immodulatory ligands and compared the effect of single agent versus combinations of checkpoint inhibitory monoclonal antibodies on T cell proliferation and cytokine production. Taqman and flow cytometry analysis was performed on several clones following allo-stimulation. A decrease in TIL clonality with repeated allo-stimulation was confirmed by TcR beta chain sequencing. In-vitro IFN gamma and Granzyme B production was enhanced with anti-PD-1 antibodies in combination with either anti-LAG3 or anti-TIGIT antibodies. This in-vitro system has the potential of screening combinations of immodulatory antibodies in a variety of TILS from different tumor types and can improve our understanding of new checkpoint inhibitor combinations. Citation Format: Robert L. Shields, Sabine Le Saux, Sophie Li, Manjiri Sathe, Terri McClanahan, Rene De Waal Malefyt. Tumor derived T cell clones for evaluation of check point inhibitor therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A33.

Abstract 963: Screening of neoantigen-specific T cells in head and neck cancer and establishment of T-cell receptor-engineered T cells with cytotoxic reactivity

Abstract Neoantigen-reactive T cells are considered to be crucial mediators for anti-tumor activity in adoptive T cell transfer (ACT) immunotherapy although the efficacy of ACT using in vitro expanded tumor-infiltrating T lymphocytes (TILs) has been limited. Since characterization of T cell receptor (TCR) repertoires of TILs before and after in vitro expansion has not well investigated, we in present study performed whole exome sequencing (WES) and transcriptome analysis, and selected candidate neoantigen epitopes to induce cytotoxic T lymphocytes (CTLs) in 20 patients with head and neck cancer (10 frozen tissues and 10 freshly resected tumors). 36 potential neoantigen peptides including missense somatic mutations were examined for induction of neoantigen-reactive cytotoxic T cells in vitro using patients' derived dendritic cells. We have so far confirmed three neoantigen-reactive T cells and obtained the sequence information of a pair of TCR alpha and beta chains, which were identical to those identified in resident dominant TILs in the original tumors. We cloned the TCR genes into T lymphocytes and constructed the neoantigen-reactive T-cell receptor-engineered (TCR-engineered) T cells, which showed HLA-restricted neoantigen-reactive cytotoxic activity. In addition, we compared the TCR composition of TILs before/after in vitro expansion in 10 fresh head and neck cancers, and found that TCR clonotypes in original TILs and in vitro expanded TILs are drastically different except for those between expanded CD8 cells and resident TILs in three tumors, which had unusually high mutational burden due to mutations in the mismatch repair genes (MSH2, MSH6) or a DNA polymerase (POLE) gene. Our data also suggest that some expanded TILs are likely to be cancer-specific T cells and may be good cell sources for ACT immunotherapy and that the mutational load might be a good biomarker to screen effective TIL treatment. We demonstrate establishment of an effective and rapid protocol to generate neoantigen-specific T cells and to identify neoantigen-specific TCRs for individual patients, which makes TCR-engineered T cells applicable for the clinical use. Citation Format: Lili Ren, Tatsuo Matsuda, Boya Deng, Kazuma Kiyotani, Taigo Kato, Jae-Hyun Park, Tanguy Seiwert, Everett Vokes, Nishant Agrawal, Yusuke Nakamura. Screening of neoantigen-specific T cells in head and neck cancer and establishment of T-cell receptor-engineered T cells with cytotoxic reactivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 963.

Abstract 1538: Analysis of the paired TCRα- and β-V(D)J full-length chains of single-cell sequence from human naïve and antigen-experienced T cells

Abstract The unbiased and paired characterization of T-cell receptor (TCR) alpha and beta chains is critical to understand the TCR repertoire and adaptive immunity. However, the direct cloning of the single human full-length TCRα- and TCRβ-chains has very few reports. We present a new methodology enabling capture of natively paired, full-length rearranged T-cell receptor (TCR) sequences at single-cell level of human T cells. More than 50% paired TCRα- and β- chains were captured in 100 single naïve human CD8+ T cells using 5'SMART and 3' TCRαβ constant region nested gene-specific primers (GSP). Furthermore, we sequenced the TCR of the specific T cells that were stimulated by representative tumor-associated antigens (TAA), virus-related antigens (VRA) and neoantigens. The highest proportion of TCR subtypes were extracted in the TCR abundance ranking by IMGT blast. Finally, we obtained the paired, full-length V(D)J TCR from melanoma antigen Melan A epitope 27-35 that was TRAV(12-2)J(47), TRBV(7-6)J(2-1), and the TCR from antigen EBV LMP2-FLY that was TRAV(26-1)J(7), TRBV(27)J(2-1). These results suggest that the method provides accurate identification of the paired, V(D)J rearrangements for each individual human naïve and antigen-experienced T cells. The information could be used in the construction of an engineered T-cell receptor for cancer immunotherapy and infectious diseases. Citation Format: Fei Wang, Qing Zhou, Cheng-chi Chao. Analysis of the paired TCRα- and β-V(D)J full-length chains of single-cell sequence from human naïve and antigen-experienced T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1538.